Spermatid Head Research Articles

Coiled-coil domain containing 159 is required for spermatid head and tail assembly in mice†.

The centrosome is critical for maintaining the sperm head-tail connection and the formation of flagellar microtubules. In this study, we found that in mouse testes, CCDC159 (coiled-coil domain-containing protein 159) is specifically localized to the head-tail coupling apparatus (HTCA) of spermatids, a structure that ensures sperm head-tail tight conjunction. CCDC159 contains a C-terminal coiled-coil domain that functions as the centrosomal localization signal. Gene knockout (KO) of Ccdc159 in mice resulted in acephalic spermatozoa, abnormal flagella, and male infertility. To explore the mechanism behind CCDC159 regulating spermatogenesis, we identified CCDC159-binding proteins using a yeast two-hybrid screen and speculated that CCDC159 participates in HTCA assembly by regulating protein phosphatase PP1 activity. Further RNA-sequencing analyses of Ccdc159 KO testes revealed numerous genes involved in male gamete generation that were downregulated. Together, our results show that CCDC159 in spermatids is a novel centrosomal protein anchoring the sperm head to the tail. Considering the limitation of KO mouse model in clarifying the biological function of CCDC159 in spermatogenesis, a gene-rescue experiment will be performed in the future.

Biallelic mutations in IQCN, encoding a novel acroplaxome protein, lead to fertilization failure and male infertility with defects in the acrosome and shaping of the spermatid head in humans and mice

Biallelic mutations in <i>IQCN</i>, encoding a novel acroplaxome protein, lead to fertilization failure and male infertility with defects in the acrosome and shaping of the spermatid head in humans and mice

Localization Patterns of RAB3C Are Associated with Murine and Human Sperm Formation.

Background and Objectives: Septins (SEPTs) are highly conserved GTP-binding proteins and the fourth component of the cytoskeleton. Polymerization of SEPTs contributes to several critical cellular processes such as cytokinesis, cytoskeletal remodeling, and vesicle transportation. In our previous study, we found that SEPT14 mutations resulted in teratozoospermia with >87% sperm morphological defects. SEPT14 interactors were also identified through proteomic assays, and one of the peptides was mapped to RAB3B and RAB3C. Most studies on the RAB3 family have focused on RAB3A, which regulates the exocytosis of neurotransmitters and acrosome reactions. However, the general expression and patterns of the RAB3 family members during human spermatogenesis, and the association between RAB3 and teratozoospermia owing to a SEPT14 mutation, are largely unknown. Materials and Methods: Human sperm and murine male germ cells were collected in this study and immunofluorescence analysis was applied on the collected sperm. Results: In this study, we observed that the RAB3C transcripts were more abundant than those of RAB3A, 3B, and 3D in human testicular tissues. During human spermatogenesis, the RAB3C protein is mainly enriched in elongated spermatids, and RAB3B is undetectable. In mature human spermatozoa, RAB3C is concentrated in the postacrosomal region, neck, and midpiece. The RAB3C signals were delocalized within human spermatozoa harboring the SEPT14 mutation, and the decreased signals were accompanied by a defective head and tail, compared with the healthy controls. To determine whether RAB3C is involved in the morphological formation of the head and tail of the sperm, we separated murine testicular tissue and isolated elongated spermatids for further study. We found that RAB3C is particularly expressed in the manchette structure, which assists sperm head shaping at the spermatid head, and is also localized at the sperm tail. Conclusions: Based on these results, we suggest that the localization of RAB3C proteins in murine and human sperm is associated with SEPT14 mutation-induced morphological defects in sperm.

Autophagy mediated tubulobulbar complex components degradation is required for spermiation

Spermiation is the process that releases mature spermatids from Sertoli cells into the lumen of the seminiferous tubule. Tubulobulbar complexes (TBCs) are elaborate cytoskeleton-related structures that are indispensable for spermiation. Despite well-defined ultrastructural events, the molecular regulation of TBCs during spermiation remains largely unknown. Here, we show that autophagy is active in TBC regions, and impaired autophagy in Sertoli cells affects spermiation. Further studies demonstrated that many TBC components bound to LC3 and could be selectively degraded through the autophagy-lysosome pathway. Perturbed autophagy impaired the degradation of some TBC components in Sertoli cells, such as VCL and CTTN, and led to the accumulation of TBC components surrounding the spermatid head, which may be associated with the sperm-releasing defect. Together, our results reveal that autophagy is essential for the TBC components degradation in mouse Sertoli cells and define a functional role of autophagy during spermiation.

The perinuclear theca protein Calicin helps shape the sperm head and maintain the nuclear structure in mice.

The perinuclear theca (PT) is a cytoskeletal element encapsulating the sperm nucleus; however, our understanding of the physiological roles of PT in sperm is very limited. We show that Calicin interacts with itself and many other PT components, indicating it may serve as an organizing center of the PT assembly. Calicin is detectable first when surrounding the acrosome, then detected around the entire nucleus, and finally translocated to the postacrosomal region of spermatid heads. Intriguingly, loss of Calicin specifically causes surface subsidence of sperm heads in the nuclear condensation stage. Calicin interacts with inner acrosomal membrane (IAM) protein Spaca1 and nuclear envelope (NE) components to form an "IAM-PT-NE" structure. Intriguingly, Ccin-knockout sperm also exhibit DNA damage and failure of fertilization. Our study provides solid animal evidence to suggest that the PT encapsulating sperm nucleus helps shape the sperm head and maintain the nuclear structure.

Lacking of sex-lethal gene lowers the fertility of male reproduction in Spodoptera litura (Lepidoptera)

Sex-lethal (Sxl) encodes an RNA-binding protein that acts as the switch of sex determination in Drosophila and influences the genitalia formation and gonadal development. However, its sex-determination roles are not conserved in all insects and its role in the gonadal development of Lepidoptera is not well documented. In this study, three splicing variants of Sxl mRNA were identified in Spodoptera litura and they highly expressed in gonads, particularly in the testis. The mRNA levels of SlSxl exhibited higher expression in the spermatid than the testis sheaths, and gradually increased with the spermiogenesis. Sex-lethal protein (SlSXL) is mainly distributed in the cytoplasm of spermatocytes and the head of spermatid. Knockout of SlSxl resulted in fewer eupyrene sperm bundles and apyrene sperm bundles in the testes of moth and a large number of undeveloped spermatocysts retained in the moth of mutant testis, and leading to the reduction of oviposition and hatch rate in the offsprings after mating with female. These results suggest that SlSxl is a critical player in the spermiogenesis of S. litura.

AXDND1, a novel testis-enriched gene, is required for spermiogenesis and male fertility

Spermiogenesis is a complex process depending on the sophisticated coordination of a myriad of testis-enriched gene regulations. The regulatory pathways that coordinate this process are not well understood, and we demonstrate here that AXDND1, as a novel testis-enriched gene is essential for spermiogenesis and male fertility. AXDND1 is exclusively expressed in the round and elongating spermatids in humans and mice. We identified two potentially deleterious mutations of AXDND1 unique to non‐obstructive azoospermia (NOA) patients through selected exonic sequencing. Importantly, Axdnd1 knockout males are sterile with reduced testis size caused by increased germ cell apoptosis and sloughing, exhibiting phenotypes consistent with oligoasthenoteratozoospermia. Axdnd1 mutated late spermatids showed head deformation, outer doublet microtubules deficiency in the axoneme, and loss of corresponding accessory structures, including outer dense fiber (ODF) and mitochondria sheath. These phenotypes were probably due to the perturbed behavior of the manchette, a dynamic structure where AXDND1 was localized. Our findings establish AXDND1 as a novel testis-enrich gene essential for spermiogenesis and male fertility probably by regulating the manchette dynamics, spermatid head shaping, sperm flagellum assembly.

Disruption of anchoring junctions in the testes of experimental varicocele rats.

Varicocele is a common disease of the male reproductive system and is the main cause of male infertility; however, the pathological mechanisms of varicocele remain unclear. The anchoring junctions (AJs) in the testies are located between Sertoli cells, or between Sertoli cells and germ cells. Intact and functional AJs are crucial for spermatogenesis. In the present study, the histomorphology, ultrastructure of AJ, cell cycle, expression of AJ structural proteins, and the level of AJ-associated signaling molecules were investigated in the left testes of experimental varicocele rats at 8 and 12 weeks after surgery. The results revealed that varicocele induced the loss of premature germ cells from the seminiferous epithelium. Furthermore, the results of the present study also revealed damage to the AJ ultrastructure, disorientation of the spermatid head, deregulation of the cell cycle, downregulation of AJ structural proteins, enhanced phosphorylation of focal adhesion kinase (FAK) at Tyr397 and its downstream adapter Src at Tyr416, and activation of the extracellular signal-regulated protein kinase 1 (ERK1) signaling pathway. Thus, the present study demonstrated that varicocele disrupted the structure and function of AJs in the left testes of rats, and that enhancement of FAK phosphorylation may contribute to AJ damage by activating ERK1 signaling, disrupting actin-based filament networks, and altering the balance of the apical ectoplasmic specialization-blood testis barrier functional axis. These findings provide important insights into the pathological mechanisms through which varicocele contributes to male infertility and could help to identify new therapeutic targets for varicocele.

WDR62 is required for centriole duplication in spermatogenesis and manchette removal in spermiogenesis

WDR62 is a scaffold protein involved in centriole duplication and spindle assembly during mitosis. Mutations in WDR62 can cause primary microcephaly and premature ovarian insufficiency. We have generated a genetrap mouse model deficient in WDR62 and characterised the developmental effects of WDR62 deficiency during meiosis in the testis. We have found that WDR62 deficiency leads to centriole underduplication in the spermatocytes due to reduced or delayed CEP63 accumulation in the pericentriolar matrix. This resulted in prolonged metaphase that led to apoptosis. Round spermatids that inherited a pair of centrioles progressed through spermiogenesis, however, manchette removal was delayed in WDR62 deficient spermatids due to delayed Katanin p80 accumulation in the manchette, thus producing misshapen spermatid heads with elongated manchettes. In mice, WDR62 deficiency resembles oligoasthenoteratospermia, a common form of subfertility in men that is characterised by low sperm counts, poor motility and abnormal morphology. Therefore, proper WDR62 function is necessary for timely spermatogenesis and spermiogenesis during male reproduction.

Bi-allelic mutation in Fsip1 impairs acrosome vesicle formation and attenuates flagellogenesis in mice

Fibrous sheath interacting protein 1 (Fsip1) is a cytoskeletal structural protein of the sperm flagellar proteome. A few studies have reported that it plays a vital role in the tumorigenesis and cancer progression. However, little is known about the role of Fsip1 in spermatogenesis and mammalian sperm flagellogenesis. Fsip1 protein showed the highest expression in round spermatids, and was translocated from nucleus to the anterior region of the elongating spermatid head. To investigate its role we constructed homozygous Fsip1 null (Fsip1−/−) mice. We found that the homozygous Fsip1−/− mutant mice were infertile, with a low sperm count and impaired motility. Interestingly, a subtle phenotype characterized by abnormal head shape, and flagella deformities was observed in the sperm of Fsip1−/− mutant mice similar to the partial globozoospermia phenotype. Electron microscopy analysis of Fsip1−/− sperm revealed abnormal accumulation of mitochondria, disrupted axoneme and retained cytoplasm. Testicular sections showed increased cytoplasmic vacuoles in the elongated spermatid of Fsip1–/–mice, which indicated an intraflagellar transport (IFT) defect. Using proteomic approaches, we characterized the cellular components and the mechanism underlying this subtle phenotype. Our result indicated that Fsip1–/–downregulates the formation of acrosomal membrane and vesicles proteins, intraflagellar transport particles B, and sperm flagellum components. Our results suggest that Fsip1 is essential for normal spermiogenesis, and plays an essential role in the acrosome biogenesis and flagellogenesis by attenuating intraflagellar transport proteins.

Tsga8 is required for spermatid morphogenesis and male fertility in mice.

During spermatogenesis, intricate gene expression is coordinately regulated by epigenetic modifiers, which are required for differentiation of spermatogonial stem cells (SSCs) contained among undifferentiated spermatogonia. We have previously found that KMT2B conveys H3K4me3 at bivalent and monovalent promoters in undifferentiated spermatogonia. Because these genes are expressed late in spermatogenesis or during embryogenesis, we expect that many of them are potentially programmed by KMT2B for future expression. Here, we show that one of the genes targeted by KMT2B, Tsga8, plays an essential role in spermatid morphogenesis. Loss of Tsga8 in mice leads to male infertility associated with abnormal chromosomal distribution in round spermatids, malformation of elongating spermatid heads and spermiation failure. Tsga8 depletion leads to dysregulation of thousands of genes, including the X-chromosome genes that are reactivated in spermatids, and insufficient nuclear condensation accompanied by reductions of TNP1 and PRM1, key factors for histone-to-protamine transition. Intracytoplasmic sperm injection (ICSI) of spermatids rescued the infertility phenotype, suggesting competency of the spermatid genome for fertilization. Thus, Tsga8 is a KMT2B target that is vitally necessary for spermiogenesis and fertility.

An actomyosin clamp assembled by the Amphiphysin-Rho1-Dia/DAAM-Rok pathway reinforces somatic cell membrane folded around spermatid heads

Membrane curvature recruits Bin-Amphiphysin-Rvs (BAR)-domain proteins and induces local F-actin assembly, which further modifies the membrane curvature and dynamics. The downstream molecular pathway invivo is still unclear. Here, we show that a tubular endomembrane scaffold supported by contractile actomyosin stabilizes the somatic cyst cell membrane folded around rigid spermatid heads during the final stages of sperm maturation in Drosophila testis. The structure resembles an actin "basket" covering the bundle of spermatid heads. Genetic analyses suggest that the actomyosin organization is nucleated exclusively by the formins - Diaphanous and Dishevelled Associated Activator of Morphogenesis (DAAM) - downstream of Rho1, which is recruited by the BAR-domain protein Amphiphysin. Actomyosin activity at the actin basket gathers the spermatid heads into a compact bundle and resists the somatic cell invasion by intruding spermatids. These observations reveal a distinct response mechanism of actin-membrane interactions, which generates a cell-adhesion-like strategy through active clamping.

Nanoscale Indentation of Plasma Membrane Establishes a Contractile Actomyosin Scaffold Through Selective Activation of the Amphiphysin-Rho1-Dia/DAAM and Rok Pathway

Nanoscale bending of plasma membrane increases cell adhesion, induces cell-signalling, triggers F-actin assembly and endocytosis in tissue-cultured cells. The underlying mechanisms are not very well understood. Here, we show that stretching the plasma membrane of somatic cyst cell around rigid spermatid heads generates a stable, tubular endomembrane scaffold supported by contractile actomyosin. The structure resembles an actin-basket covering the bundle of spermatid heads. Genetic analysis suggests that the actomyosin organisation is nucleated exclusively by the Formins, Diaphanous and DAAM, downstream of Rho1, recruited by the Bin-Amphyphysin-Rvs (BAR)-domain protein, Amphiphysin, around the spermatid heads. Actomyosin activity at the actin-basket gathers the spermatid heads into a compact bundle and resists the invasion of the somatic cell by the intruding spermatids. These observations revealed a new response mechanism of nanoscale bending of the plasma membrane, which generates a novel cell adhesion strategy through active clamping.

Knockout of family with sequence similarity 170 member A (Fam170a) causes male subfertility, while Fam170b is dispensable in mice†.

Families with sequence similarity 170 members A and B (FAM170A and FAM170B) are testis-specific, paralogous proteins that share 31% amino acid identity and are conserved throughout mammals. While previous in vitro experiments suggested that FAM170B, an acrosome-localized protein, plays a role in the mouse sperm acrosome reaction and fertilization, the role of FAM170A in the testis has not been explored. In this study, we used CRISPR/Cas9 to generate null alleles for each gene, and homozygous null (−/−) male mice were mated to wild-type females for 6 months to assess fertility. Fam170b−/− males were found to produce normal litter sizes and had normal sperm counts, motility, and sperm morphology. In contrast, mating experiments revealed significantly reduced litter sizes and a reduced pregnancy rate from Fam170a−/− males compared with controls. Fam170a−/−;Fam170b−/− double knockout males also produced markedly reduced litter sizes, although not significantly different from Fam170a−/− alone, suggesting that Fam170b does not compensate for the absence of Fam170a. Fam170a−/− males exhibited abnormal spermiation, abnormal head morphology, and reduced progressive sperm motility. Thus, FAM170A has an important role in male fertility, as the loss of the protein leads to subfertility, while FAM170B is expendable. The molecular functions of FAM170A in spermatogenesis are as yet unknown; however, the protein localizes to the nucleus of elongating spermatids and may mediate its effects on spermatid head shaping and spermiation by regulating the expression of other genes. This work provides the first described role of FAM170A in reproduction and has implications for improving human male infertility diagnoses.

Cellular Evidence of CD63-Enriched Exosomes and Multivesicular Bodies within the Seminiferous Tubule during the Spermatogenesis of Turtles.

The seminiferous tubule (ST) is the location of spermatogenesis, where mature spermatozoa are produced with the assistance of Sertoli cells. The role of extracellular vesicles in the direct communication between Sertoli-germ cells in the ST is still not fully understood. In this study, we reported multivesicular bodies (MVBs) and their source of CD63-enriched exosomes by light and ultrastructure microscopy during the reproductive phases of turtles. Strong CD63 immunopositivity was detected at the basal region in the early and luminal regions of the ST during late spermatogenesis by immunohistochemistry (IHC), immunofluorescence (IF), and western blot (WB) analysis. Labeling of CD63 was detected in the Sertoli cell cytoplasmic processes that surround the developing germ cells during early spermatogenesis and in the lumen of the ST with elongated spermatids during late spermatogenesis. Furthermore, ultrastructure analysis confirmed the existence of numerous MVBs in the Sertoli cell prolongations that surround the round and primary spermatogonia during acrosome biogenesis and with the embedded heads of spermatids in the cytoplasm of Sertoli cells. Additionally, in spermatids, Chrysanthemum flower centers (CFCs) generated isolated membranes involved in MVBs and autophagosome formation, and their fusion to form amphiosomes was also observed. Additionally, autophagy inhibition by 3-methyladenine (after 24 h) increased CD63 protein signals during late spermatogenesis, as detected by IF and WB. Collectively, our study found MVBs and CD63 rich exosomes within the Sertoli cells and their response to autophagy inhibition in the ST during the spermatogenesis in the turtle.

Proteomic alterations underlie an association with teratozoospermia in obese mice sperm

BackgroundObesity is a worldwide crisis impairing human health. In this condition, declines in sperm quality stem from reductions in sperm concentration, motility and increase in sperm deformity. The mechanism underlying these alterations remains largely unknown. This study, determined if obesity-associated proteomic expression patterns in mice sperm parallel those in spermatozoa obtained from obese humans.MethodsAn obese mouse model was established via feeding a high-fat diet (HFD). Histological analysis identified testicular morphology and a computer assisted semen analyzer (CASA) evaluated sperm parameters. Proteome analysis was performed using a label-free quantitative LC-MS/MS system. Western blot, immunohistochemical and immunofluorescent analyses characterized protein expression levels and localization in testis, sperm and clinical samples.ResultsBodyweight gains on the HFD induced hepatic steatosis. Declines in sperm motility accompanied sperm deformity development. Differential proteomic analysis identified reduced cytoskeletal proteins, centrosome and spindle pole associated protein 1 (CSPP1) and Centrin 1 (CETN1), in sperm from obese mice. In normal weight mice, both CSPP1 and CETN1 were localized in the spermatocytes and spermatids. Their expression was appreciable in the post-acrosomal region parallel to the microtubule tracks of the manchette structure in spermatids, which affects spermatid head shaping and morphological maintenance. Moreover, CSPP1 was localized in the head–tail coupling apparatus of the mature sperm, while CETN1 expression was delimited to the post-acrosomal region within the sperm head. Importantly, sperm CSPP1 and CETN1 abundance in both the overweight and obese males decreased in comparison with that in normal weight men.ConclusionThese findings show that regionally distinct expression and localization of CETN1 and CSPP1 is strongly related to spermiogenesis and sperm morphology maintaining. Obesity is associated with declines in the CETN1 and CSPP1 abundance and compromise of both sperm morphology in mice and relevant clinical samples. This parallelism between altered protein expression in mice and humans suggests that these effects may contribute to poor sperm quality including increased deformity.

Testicular Structure and Spermatogenesis in the Naked Mole-Rat Is Unique (Degenerate) and Atypical Compared to Other Mammals.

The naked mole-rat (NMR) queen controls reproduction in her eusocial colony by usually selecting one male for reproduction and suppressing gametogenesis in all other males and females. Simplified, polymorphic and slow-swimming spermatozoa in the NMR seem to have been shaped by a low risk of sperm competition. We hypothesize that this unique mammalian social organization has had a dramatic influence on testicular features and spermatogenesis in the NMR. The testicular structure as well as spermatogenic cell types and its organization in breeding, subordinate and disperser males were studied using light microscopy and transmission electron microscopy. Even though the basic testicular design in NMRs is similar to most Afrotheria as well as some rodents with intra-abdominal testes, the Sertoli and spermatogenic cells have many atypical mammalian features. Seminiferous tubules are distended and contain large volumes of fluid while interstitial tissue cover about 50% of the testicular surface area and is mainly composed of Leydig cells. The Sertoli cell cytoplasm contains an extensive network of membranes and a variety of fluid-containing vesicles. Furthermore, Sertoli cells form numerous phagosomes that often appear as extensive accumulations of myelin. Another unusual feature of mature NMR Sertoli cells is mitotic division. While the main types of spermatogonia and spermatocytes are clearly identifiable, these cells are poorly organized and many spermatids without typical intercellular bridges are present. Spermatid heads appear to be malformed with disorganized chromatin, nuclear fragmentation and an ill-defined acrosome formed from star-like Golgi bodies. Rudimentary manchette development corresponds with the occurrence of abnormal sperm morphology. We also hypothesize that NMR testicular organization and spermiation are modified to produce spermatozoa on demand in a short period of time and subsequently use a Sertoli cell “pump” to flush the spermatozoa into short tubuli recti and simplified rete testis. Despite the difficulty in finding cellular associations during spermatogenesis, six spermatogenic stages could be described in the NMR. These numerous atypical and often simplified features of the NMR further supports the notion of degenerative orthogenesis that was selected for due to the absence of sperm competition.

The perforatorium and postacrosomal sheath of rat spermatozoa share common developmental origins and protein constituents†.

The perinuclear theca (PT) is a cytosolic protein capsule that surrounds the nucleus of eutherian spermatozoa. Compositionally, it is divided into two regions: the subacrosomal layer (SAL) and the postacrosomal sheath (PAS). In falciform spermatozoa, a third region of the PT emerges that extends beyond the nuclear apex called the perforatorium. The formation of the SAL and PAS differs, with the former assembling early in spermiogenesis concomitant with acrosome formation, and the latter dependent on manchette descent during spermatid elongation. The perforatorium also forms during the elongation phase of spermiogenesis, suggesting that like the PAS, its assembly is facilitated by the manchette. The temporal similarity in biogenesis between the PAS and perforatorium led us to compare their molecular composition using cell fractionation and immunodetection techniques. Although the perforatorium is predominantly composed of its endemic protein FABP9/PERF15, immunolocalization indicates that it also shares proteins with the PAS. These include WBP2NL/PAWP, WBP2, GSTO2, and core histones, which have been implicated in early fertilization and zygotic events. The compositional homogeny between the PAS and perforatorium supports our observation that their development is linked. Immunocytochemistry indicates that both PAS and perforatorial biogenesis depend on the transport and deposition of cytosolic proteins by the microtubular manchette. Proteins translocated from the manchette pass ventrally along the spermatid head into the apical perforatorial space prior to PAS deposition in the wake of manchette descent. Our findings demonstrate that the perforatorium and PAS share a mechanism of developmental assembly and thereby contain common proteins that facilitate fertilization.

Small RNAs in Rat Sperm Are a Predictive and Sensitive Biomarker of Exposure to the Testicular Toxicant Ethylene Glycol Monomethyl Ether.

Testicular histology and semen parameters are considered the gold standards when determining male reproductive toxicity. Ethylene glycol monomethyl ether (EGME) is a testicular toxicant with well-described effects on histopathology and sperm parameters. To compare the predictivity and sensitivity of molecular biomarkers of testicular toxicity to the traditional endpoints, small RNAs in the sperm were analyzed by next generation RNA-sequencing (RNA-seq). Adult rats were exposed to 0, 50, 60, or 75 mg/kg EGME by oral gavage for 5 consecutive days. Testis histology, epididymal sperm motility, and sperm small RNAs, including microRNAs (miRNAs), mRNA fragments, piwi-interacting RNAs (piRNAs), and tRNA fragments (tRFs), were analyzed 5 weeks after cessation of exposure. Testicular histology showed a significant dose-dependent increase in retained spermatid heads (RSH), while sperm motility declined with increasing dose. RNA-sequencing of sperm small RNAs was used to identify significant dose-dependent changes in percent mRNA fragments (of total reads), percent miRNAs (of total reads), average tRF length, average piRNA length, and piRNA and tRF length-distributions. Discriminant analysis showed relatively low predictivity of exposure based on RSH or motility compared to the average read length of all assigned RNAs. Benchmark dose (BMD) modeling resulted in a BMD of 62 mg/kg using RSH, whereas average read length of all assigned RNAs resulted in a BMD of 47 mg/kg. These results showed that sperm small RNAs are sensitive and predictive biomarkers of EGME-induced male reproductive toxicity.

Vangl2 regulates spermatid planar cell polarity through microtubule (MT)-based cytoskeleton in the rat testis

During spermatogenesis, developing elongating/elongated spermatids are highly polarized cells, displaying unique apico-basal polarity. For instance, the heads of spermatids align perpendicular to the basement membrane with their tails pointing to the tubule lumen. Thus, the maximal number of spermatids are packed within the limited space of the seminiferous epithelium to support spermatogenesis. Herein, we reported findings that elongating/elongated spermatids displayed planar cell polarity (PCP) in adult rat testes in which the proximal end of polarized spermatid heads were aligned uniformly across the plane of the seminiferous epithelium based on studies using confocal microscopy and 3-dimensional (D) reconstruction of the seminiferous tubules. We also discovered that spermatid PCP was regulated by PCP protein Vangl2 (Van Gogh-like protein 2) since Vangl2 knockdown by RNAi was found to perturb spermatid PCP. More important, Vangl2 exerted its regulatory effects through changes in the organization of the microtubule (MT)-based cytoskeleton in the seminiferous epithelium. These changes were mediated via the downstream signaling proteins atypical protein kinase C ξ (PKCζ) and MT-associated protein (MAP)/microtubule affinity-regulating kinase 2 (MARK2). These findings thus provide new insights regarding the biology of spermatid PCP during spermiogenesis.